Previous work on this project has confirmed the prediction that the ratios of the adenine nucleotides, conveniently expressed by the parameter adenylate energy charge, are more important for metabolic regulation than are concentrations of individual nucleotides, that the value of the charge is held within narrow limits in vivo, and that the value of the charge is a factor in the regulation of most, if not all, metabolic sequences. We will test the effect of energy charge in the phosphofructokinase-fructose diphosphatase-aldolase region of metabolism (glycolysis and gluconeogenesis) and study regulation of the pentose phosphate pathway. Regulation of adenylate synthesis at the adenylosuccinate synthase step will be studied. We will attempt to correlate regulation of enzymes that use GTP, UTP, and CTP as phosphoryl donors with the general adenylate controls of the cell. The results of ligand binding studies, initially on phosphofructokinase, will be compared with kinetic parameters of regulatory enzymes. We intend to initiate a study of primary nitrogen metabolism, focussing on incorporation of ammonia, and to seek evidence of energy charge control of this process.